Crystallographic data of an importin-α3 dimer in which the two protomers are bridged by a bipartite nuclear localization signal

53BP1 (TP53-binding protein 1), a key player in DNA double-strand break repair, has a classical bipartite nuclear localization signal (NLS) of sequence 1666-GKRKLITSEEERSPAKRGRKS-1686 that binds to importin-α, a nuclear import adaptor protein. Nucleoporin Nup153 is involved in nuclear import of 53BP1, and the binding of Nup153 to importin-α has been proposed to promote efficient import of classical NLS-containing proteins. Here, the ARM-repeat domain of human importin-α3 bound to 53BP1 NLS was crystallized in the presence of a synthetic peptide corresponding to the extreme C-terminus of Nup153 (sequence: 1459-GTSFSGRKIKTAVRRRK-1475). The crystal belonged to space group I2, with unit-cell parameters a = 95.70, b = 79.60, c = 117.44 Å, β = 95.57°. The crystal diffracted X-rays to 1.9 Å resolution, and the structure was solved by molecular replacement. The asymmetric unit contained two molecules of importin-α3 and two molecules of 53BP1 NLS. Although no convincing density was observed for the Nup153 peptide, the electron density corresponding to 53BP1 NLS was unambiguous and continuous along the entire length of the bipartite NLS. The structure revealed a novel dimer of importin-α3, in which two protomers of importin-α3 are bridged by the bipartite NLS of 53BP1. In this structure, the upstream basic cluster of the NLS is bound to the minor NLS-binding site of one protomer of importin-α3, whereas the downstream basic cluster of the same chain of NLS is bound to the major NLS-binding site of another protomer of importin-α3. This quaternary structure is distinctly different from the previously determined crystal structure of mouse importin-α1 bound to the 53BP1 NLS. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession code 8HKW).

a b s t r a c t 53BP1 (TP53-binding protein 1), a key player in DNA double-strand break repair, has a classical bipartite nuclear localization signal (NLS) of sequence 1666-GKRKLITSEEERSPAKRGRKS-1686 that binds to importin-α, a nuclear import adaptor protein. Nucleoporin Nup153 is involved in nuclear import of 53BP1, and the binding of Nup153 to importin-α has been proposed to promote efficient import of classical NLS-containing proteins. Here, the ARM-repeat domain of human importin-α3 bound to 53BP1 NLS was crystallized in the presence of a synthetic peptide corresponding to the extreme C-terminus of Nup153 (sequence: 1459-GTSFSGRKIKTAVRRRK-1475). The crystal belonged to space group I 2, with unit-cell parameters a = 95.70, b = 79.60, c = 117.44 Å , β = 95.57 °. The crystal diffracted X-rays to 1.9 Å resolution, and the structure was solved by molecular replacement. The asymmetric unit contained two molecules of importin-α3 and two molecules of 53BP1 NLS. Although no convincing density was observed for the Nup153 peptide, the electron density corresponding to 53BP1 NLS was unambiguous and continuous along the entire length of the bipartite NLS. The structure revealed a novel dimer of importin-α3, in which two protomers of importin-α3 are bridged by the bipartite NLS of 53BP1.
In this structure, the upstream basic cluster of the NLS is bound to the minor NLS-binding site of one protomer of importin-α3, whereas the downstream basic cluster of the same chain of NLS is bound to the major NLS-binding site of another protomer of importin-α3. This quaternary structure is distinctly different from the previously determined crystal structure of mouse importin-α1 bound to the 53BP1 NLS.

Value of the Data
• The significance of importin-α oligomerization has been debated in nuclear transport field. The data reported here extends our knowledge on importin-α oligomerization and will benefit researchers studying the structure and function of nuclear transport receptors. • The purification procedure, crystallization condition, and a photograph of the crystal used in this work serve as a useful reference for researchers interested in structural study of importin-α-cargo complexes.
• Although the quaternary structure reported here could be an artifact of the crystallization process, the data enriches structural database of what can happen during crystallization of protein-peptide complexes, and will benefit researchers interested in protein crystallization. • The molecular mechanism of interactions between nucleoporins and nuclear transport receptors is not fully understood, and remains an important issue in nuclear transport field. The data that convincing electron density corresponding to Nup153 was not observed in the crystal structure reported here is a negative data useful for researchers studying nucleoporin-importin interactions.

Objective
The initial objective of this work was to gain structural insights into how Nup153 interacts with importin-α and promotes nuclear import of NLS-containing proteins such as 53BP1. However, this work serendipitously led to a discovery of a novel quaternary structure of importin-α-NLS complexes that can be formed at least in the crystal.

Data Description
X-ray diffraction dataset up to 1.9 Å resolution was collected from a single crystal of the ARM-repeat domain of human importin-α3 bound to the bipartite NLS of 53BP1 (human 53BP1 residues 1665-1686; [2] ), grown in the presence of a Nup153 peptide (referred to as Nup153C), at the Photon Factory beamline BL-17A. The Nup153C peptide corresponds to the extreme Cterminus of human Nup153 (residues 1459-1475). Previous studies have shown that Nup153 is involved in nuclear import of 53BP1 [3] and that the interaction between Nup153 and importinα is required for efficient nuclear import of classical NLS-containing proteins [4] . It has been proposed that the extreme C-terminus of Nup153 binds directly to importin-α [ 4 , 5 ]. Fig. 1 shows the plate-shaped crystal used for data collection. The structure was solved by molecular replacement, with the structure of importin-α3 bound to Hendra virus W protein Cterminus (PDB code, 6BW9) [6] as a search model. The structure was refined to free and working R -factor values of 22.14% and 20.06%, respectively. Fig. 2 shows the overall structure of the asymmetric unit, which contained two molecules of importin-α3 and two molecules of the 53BP1 NLS. Data collection and refinement statistics are shown in Table 1 .  As shown in Fig. 2 , the 53BP1 NLS was unambiguously identified in the electron density map, and the density was continuous along the entire length of this bipartite NLS. However, no convincing density was observed for the Nup153C peptide. This quaternary structure of importin-α3-53BP1 NLS complex is distinctly different from the previously reported crystal structure of mouse importin-α1-53BP1 NLS complex, in which one molecule of 53BP1 NLS is bound along the concave surface of one molecule of importin-α1 [7] .

Crystallization, data collection, and structure determination
A synthetic peptide (referred to as Nup153C) corresponding to the extreme C-terminus (residues 1459-1475) of human Nup153 ( 1459 GTSFSGRKIKTAVRRRK 1475 ) was synthesized by Gen-Script. A plate-shaped crystal was obtained using hanging drop vapor diffusion at 20 °C with 1.5 μl of a protein solution (0.58 mM importin-α3-53BP1 NLS complex and 0.71 mM Nup153C in buffer C) mixed with 1.5 μl of mother liquor (0.2 M LiNO 3 and 25% PEG3350) over a reservoir of 600 μl of mother liquor. The crystal was briefly soaked in a cryoprotection solution (0.2 M LiNO 3 , 28% PEG3350, 6% PEG400, and 0.5 mM Nup153C) for less than 30 s, and flash-cooled in liquid nitrogen. X-ray diffraction datasets were collected at 95 K at the Photon Factory beamline BL-17A using an EIGER X 16 M detector at a wavelength of 0.98 Å . Diffraction data were processed using iMOSFLM and CCP4 programs [9] . The structure was solved by molecular replacement using MOLREP [10] , with the structure of importin-α3 bound to Hendra virus W protein C-terminus (PDB code, 6BW9) [6] as a search model. The atomic model was iteratively rebuilt using COOT [11] and refined using PHENIX [12] . MolProbity [13] was used to validate the final model. CCP4MG [14] was used to generate structural figures. The atomic coordinates and structure factors have been deposited in the PDB with accession code 8HKW [1] .

Ethics Statements
This work meets the ethical requirements for publication in this journal. This work does not involve human subjects, animal experiments, or any data collected from social media.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Data availability
Crystal structure of importin-alpha3 bound to the 53BP1 nuclear localization signal (Original data) (Protein Data Bank).